EP0473328A2 - Biological process for preparing optically active lactic acid - Google Patents
Biological process for preparing optically active lactic acid Download PDFInfo
- Publication number
- EP0473328A2 EP0473328A2 EP91307462A EP91307462A EP0473328A2 EP 0473328 A2 EP0473328 A2 EP 0473328A2 EP 91307462 A EP91307462 A EP 91307462A EP 91307462 A EP91307462 A EP 91307462A EP 0473328 A2 EP0473328 A2 EP 0473328A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- ferm
- pseudomonas
- lactic acid
- rhodococcus
- arthrobacter
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
Definitions
- the invention relates to a process for preparing an optically active lactic acid from DL-lactonitrile using a microorganism.
- An optically active lactic acid is useful as a chiral center for producing optically active pharmaceuticals and agricultural chemicals.
- D-lactic acid is expected to enjoy an increasing demand as a starting material for L-2-chloropropionic acid, an intermediate of optically active herbicides.
- L-2-chloropropionic acid an intermediate of optically active herbicides.
- much study is directed to the use of optically active lactic acid polymers as medical materials.
- Known processes for biologically preparing lactic acid from DL-lactonitrile include a process of using a microorganism belonging to the genus Bacillus , Bacteridium , Micrococcus or Brevibacterium (see U.S.
- Patent 3,940,316 a process of using a microorganism belonging to the genus Corynebacterium (see JP-A-61-56086, the term "JP-A” as used herein means an "unexamined published Japanese patent application”); a process of using a microorganism belonging to the genus Corynebacterium , Nocardia , Bacillus , Bacteridium , Micrococcus or Brevibacterium (see JP-A-61-162191); and a process of using a microorganism belonging to the genus Pseudomonas , Arthrobacter , Aspergillus , Penicillium , Cochliobolus or Fusarium (see JP-A-63-222696). Those processes relate to production of racemic lactic acid and not to production of an optically active lactic acid.
- an object of the present invention is to provide an industrially advantageous process for preparing an optically active lactic acid from DL-lactonitrile.
- an optically active lactic acid can be obtained efficiently by subjecting DL-lactonitrile to the action of a specific microorganism capable of asymmetrically hydrolyzing a nitrile in a polar solvent.
- the inventors have ascertained that lactonitrile has the property of coming to equilibrium upon dissociation into acetaldehyde and prussic acid in a polar solvent, which eventually leads to racemization.
- a desired optically active lactic acid can be prepared in a predominant proportion directly from DL-lactonitrile by combining such a racemization system with a nitrile asymmetric hydrolase produced from a microorganism. It is possible theoretically to convert all the starting materials to a desired optically active lactic acid.
- the present invention has been completed based on those findings.
- the present invention relates to a process for preparing an optically active lactic acid comprising subjecting DL-lactonitrile to the action of a microorganism belonging to the genus Enterobacter , Arthrobacter , Caseobacter , Brevibacterium , Aureobacterium , Escherichia , Micrococcus , Streptomyces , Flavobacterium , Aeromonas , Nocardia , Mycoplana , Cellulomonas , Erwinia , Candida , Pseudomonas , Rhodococcus , Bacillus , Alcaligenes , Corynebacterium , Microbacterium or Obsumbacterium , or treated microbial cells thereof, which said microorganism is capable of stereospecifically hydrolyzing a nitrile in a polar solvent to produce directly D-lactic acid or L-lactic acid in a predominant proportion to said starting DL-actonitrile.
- the DL-lactonitrile starting material is replaced by prussic acid and acetaldehyde.
- the microorganism used in the present invention is a microorganism belonging to the genus Enterobacter , Arthrobacter , Caseobacter , Brevibacterium , Aureobacterium , Escherichia , Micrococcus , Streptomyces , Flavobacterium , Aeromonas , Nocardia , Mycoplana , Cellulomonas , Erwinia , Candida , Pseudomonas , Rhodococcus , Bacillus , Alcaligenes , Corynebacterium , Microbacterium or Obsumbacterium , which is capable of stereospecifically hydrolyzing a nitrile.
- Those microorganisms can be obtained, for example, by the following process.
- the soil collected at Yokohama-shi of Kanagawa, Japan is suspended in 15 ml of a sterilized water and allowed to stand for 30 minutes. Then, 0.1 to 0.2 ml of the resulting supernatant is sprayed on an agar medium containing 2-chloropropionitrile as a nitrogen source (20 g of glycerol, 1 g of 2-chloropropionitrile, 3 g of K2HPO4, 0.2 g of MgCl2, 40 mg of CaCl2, 4 mg of MnSO4.4H2O, 0.7 mg of FeCl3.7H2O, 0.1 mg of ZnSO4.7H2O and 15 g of agar in 1 l of demineralized water (pH 7.0) and then cultured at 30°C for 5 to 6 days. The colonies formed are purely isolated on the agar medium.
- the thus-obtained strains are cultured in the same manner as in Example 1 below and reacted with a lactonitrile. After completion of the reaction, the system is subjected to centrifugation to remove the microbial cells. The supernatant liquor is analyzed by liquid chromatography (column: SHODEX ODS F511A, carrier: 0.2M H3PO2, column temp.: 30°C, monitor wavelength: 208 nm) to determine an amount of lactic acid produced.
- P-11311 Pseudomonas sp. BC13-2 (FERM No. BP-3319), Pseudomonas sp. BC15-2 (FERM No. BP-3320), Pseudomonas synxantha IAM 12356, Pseudomonas aeruginosa IAM 1267, Pseudomonas ovalis IAM 1002, Rhodococcus erythropolis IFO 12540, Rhodococcus erythropolis IFO 12320, Rhodococcus sp. SK92 (FERM No. BP-3324), Rhodococcus sp. HR11 (FERM No. P-11306), Rhodococcus sp.
- SK70 (FERM No. P-11304), Rhodococcus erythropolis IFO 12538, Rhodococcus erythropolis IFO 12539, Rhodococcus erythropolis IFM 132, Rhodococcus erythropolis IFM 155, Rhodococcus rhodochrous ATCC 12674, Bacillus licheniformis IFO 12197, Bacillus licheniformis IFO 12199, Bacillus megaterium ATCC 25833, Bacillus subtilis ATCC 21697, Alcaligenes sp. BC20 (FERM No. P-11264), Corynebacterium flavescens IAM 1642, Microbacterium lacticum IAM 1640 and Obsumbacterium proteus ATCC 12841, and variants of those strains.
- microorganisms are new strains isolated from nature by the present inventors and have been deposited with the Fermentation Research Institute, Agency of Industrial Science & Technology under the respective deposit number (FERM Nos.) listed above. The morphologic and physiologic properties of the new strains are described below.
- the SK12 strain was identified to belong to the genus Enterobacter ; the SK103 and HR1 strains to the genus Arthrobacter ; the BC4 and BC23 strains to the genus Caseobacter ; the SK150 strain to the genus Flavobacterium ; the SK13, SK31, SK87, BC13-2 and BC15-2 strains to the genus Pseudomonas ; the SK92, HR11 and SK70 strains to the genus Rhodococcus ; and the BC20 strain to the genus Alcaligenes , respectively.
- Culture media which can be used for culturing the microorganisms according to the present invention usually contain assimilable carbon sources and nitrogen sources and inorganic nutrients necessary for growth.
- Carbon sources include glucose, glycerol and sucrose.
- Nitrogen sources include yeast extract, peptone and ammonium sulfate.
- Inorganic nutrients include dipotassium hydrogenphosphate, magnesium chloride and ferric chloride.
- nitriles e.g., 2-chloropropionitrile, acetonitrile, propionitrile, n-butyronitrile, isobutyronitrile, benzonitrile, benzyl cyanide and 3-cyanopyridine
- amides or lactams corresponding to those nitriles e.g., ⁇ -caprolactam and ⁇ -butyrolactam
- Culturing is conducted aerobically under controlled conditions selected according to the microorganism, i.e., at a pH of from 4 to 10 and at a temperature of from 20 to 90°C for a period of from 24 to 96 hours.
- the hydrolysis of DL-lactonitrile is carried out by bringing DL-lactonitrile into contact with the thus-obtained microbial cells or treated microbial cells (ruptured cells, crude or purified enzyme isolated therefrom, immobilized microbial cells or enzyme) in a polar solvent, whereby a desired optically active lactic acid, i.e., D- or L-lactic acid, is obtained from DL-lactonitrile in a predominant proportion (i.e., 50 to 100%) and in a high yield.
- Typical examples of usable polar solvents include water and an aqueous medium, e.g., physiologic saline and a phosphoric acid buffer solution.
- organic solvents such as lower alcohols (e.g., methanol and ethanol), dimethylformamide and dioxane, may be used in combination with the polar solvent.
- the hydrolysis reaction also can be carried out by using acetaldehyde and prussic acid in place of DL-lactonitrile.
- DL-Lactonitrile usually is used at a concentration of from 0.01 to 10% by weight in the reaction system and the concentrations of acetaldehyde and prussic acid are 0.01 to 5% by weight and 0.005 to 3% by weight, respectively.
- the microbial cells usually are used in an amount of from 0.01 to 5% by weight (dry basis) based on DL-lactonitrile.
- the reaction is performed at a pH of from 3 to 12, and preferably from 6 to 10, and at a temperature of from the freezing point to 70°C, and preferably from 10 to 50°C for a period of from 0.5 to 72 hours.
- the desired optically active lactic acid is isolated from the reaction solution by any of known techniques, such as concentration, electrical dialysis, ion exchange, extraction and crystallization.
- a desired optically active lactic acid can be prepared directly from DL-lactonitrile in a predominant proportion (50 to 100%).
- the present invention makes it feasible to convert stoichiometrically all the raw material to a desired optically active lactic acid, thus providing an extremely efficient process for preparing optically active lactic acids.
- Each of the microorganisms shown in Table 1 below was inoculated into 10 ml of a medium having the following composition in a test tube (diameter: 22 mm) and shake-cultured at 30°C for 2 days.
- Enterobacter sp. SK12 was cultured in the same manner as in Example 1, except for using 100 ml of the medium in a 500 ml volume Erlenmeyer flask.
- Example 2 To the microbial cells collected from the culture in the same manner as in Example 1 was added 50 mM phosphoric acid buffer solution to prepare a cell suspension having a cell concentration of 30 (OD 630 nm). To the cell suspension was added DL-lactonitrile to a final concentration of 20 mM, and the reaction progress was observed over time. A 2 ml sample of the reaction system was taken out each 1 hour to determine the amount of lactic acid produced in the same manner as in Example 1. The results obtained are shown in Table 2 below.
- Enterobacter sp. SK12 was cultured in the same manner as in Example 2.
- Example 2 To the microbial cells collected from the culture in the same manner as in Example 2 was added 50 mM phosphoric acid buffer solution to prepare a cell suspension having a cell concentration of 30 (OD 630 nm). To the cell syspension was added acetaldehyde and prussic acid to a final concentration of 20 mM, respectively, and the reaction was carried out at 30°C for 5 hours. The amount of lactic acid produced and its optical purity were determined in the same manner as in Example 1. The results obtainedare shown in the Table 3 below.
Abstract
A process for preparing an optically active lactic acid comprising subjecting DL-lactonitrile or acetaldehyde and prussic acid to the action of a microorganism belonging to the genus Enterobacter, Arthrobacter, Caseobacter, Brevibacterium, Aureobacterium, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Nocardia, Mycoplana, Cellulomonas, Erwinia, Candida, Pseudomonas, Rhodococcus, Bacillus, Alcaligenes, Corynebacterium, Microbacterium or Obsumbacterium, or treated microbial cells thereof, which the microorganism is capable of stereospecifically hydrolyzing a nitrile in a polar solvent. D-Lactic acid or L-lactic acid can be produced directly in a predominant proportion from the starting materials.
Description
- The invention relates to a process for preparing an optically active lactic acid from DL-lactonitrile using a microorganism. An optically active lactic acid is useful as a chiral center for producing optically active pharmaceuticals and agricultural chemicals. For example, D-lactic acid is expected to enjoy an increasing demand as a starting material for L-2-chloropropionic acid, an intermediate of optically active herbicides. Further, increasingly, much study is directed to the use of optically active lactic acid polymers as medical materials.
- Known processes for biologically preparing lactic acid from DL-lactonitrile include a process of using a microorganism belonging to the genus Bacillus, Bacteridium, Micrococcus or Brevibacterium (see U.S. Patent 3,940,316); a process of using a microorganism belonging to the genus Corynebacterium (see JP-A-61-56086, the term "JP-A" as used herein means an "unexamined published Japanese patent application"); a process of using a microorganism belonging to the genus Corynebacterium, Nocardia, Bacillus, Bacteridium, Micrococcus or Brevibacterium (see JP-A-61-162191); and a process of using a microorganism belonging to the genus Pseudomonas, Arthrobacter, Aspergillus, Penicillium, Cochliobolus or Fusarium (see JP-A-63-222696). Those processes relate to production of racemic lactic acid and not to production of an optically active lactic acid.
- On the other hand, with respect to production of optically active hydroxy acids, there are known a process for producing an L-α-hydroxy acid which uses yeast of the genus Torulopsis (see JP-B-54-14668, the term "JP-B" as used herein means an "examined published Japanese patent application") and a process for producing an optically active α-substituted organic acid which uses a microorganism belonging to the genus Alcaligenes, Pseudomonas, Rhodopsuedomonas, Corynebacterium, Acinetobacter, Bacillus, Mycobacterium, Rhodococcus or Candida (see EP 0348901A). However, those processes do not make it possible to obtain a desired optically active compound in a predominant proportion from a starting racemate. Further, these are no illustrative examples concerning the preparation of an optically active lactic acid from DL-lactonitrile and thus, it is unclear whether an optically active lactic acid can be prepared with high efficiency and at a high optical purity by those processes.
- Accordingly, an object of the present invention is to provide an industrially advantageous process for preparing an optically active lactic acid from DL-lactonitrile.
- The inventors have conducted extensive investigation into that problem. As a result, they have found that an optically active lactic acid can be obtained efficiently by subjecting DL-lactonitrile to the action of a specific microorganism capable of asymmetrically hydrolyzing a nitrile in a polar solvent. The inventors have ascertained that lactonitrile has the property of coming to equilibrium upon dissociation into acetaldehyde and prussic acid in a polar solvent, which eventually leads to racemization. Thus, a desired optically active lactic acid can be prepared in a predominant proportion directly from DL-lactonitrile by combining such a racemization system with a nitrile asymmetric hydrolase produced from a microorganism. It is possible theoretically to convert all the starting materials to a desired optically active lactic acid. The present invention has been completed based on those findings.
- The present invention relates to a process for preparing an optically active lactic acid comprising subjecting DL-lactonitrile to the action of a microorganism belonging to the genus Enterobacter, Arthrobacter, Caseobacter, Brevibacterium, Aureobacterium, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Nocardia, Mycoplana, Cellulomonas, Erwinia, Candida, Pseudomonas, Rhodococcus, Bacillus, Alcaligenes, Corynebacterium, Microbacterium or Obsumbacterium, or treated microbial cells thereof, which said microorganism is capable of stereospecifically hydrolyzing a nitrile in a polar solvent to produce directly D-lactic acid or L-lactic acid in a predominant proportion to said starting DL-actonitrile.
- In another embodiment, the DL-lactonitrile starting material is replaced by prussic acid and acetaldehyde.
- The microorganism used in the present invention is a microorganism belonging to the genus Enterobacter, Arthrobacter, Caseobacter, Brevibacterium, Aureobacterium, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Nocardia, Mycoplana, Cellulomonas, Erwinia, Candida, Pseudomonas, Rhodococcus, Bacillus, Alcaligenes, Corynebacterium, Microbacterium or Obsumbacterium, which is capable of stereospecifically hydrolyzing a nitrile. Those microorganisms can be obtained, for example, by the following process.
- The soil collected at Yokohama-shi of Kanagawa, Japan is suspended in 15 ml of a sterilized water and allowed to stand for 30 minutes. Then, 0.1 to 0.2 ml of the resulting supernatant is sprayed on an agar medium containing 2-chloropropionitrile as a nitrogen source (20 g of glycerol, 1 g of 2-chloropropionitrile, 3 g of K₂HPO₄, 0.2 g of MgCl₂, 40 mg of CaCl₂, 4 mg of MnSO₄.4H₂O, 0.7 mg of FeCl₃.7H₂O, 0.1 mg of ZnSO₄.7H₂O and 15 g of agar in 1 ℓ of demineralized water (pH 7.0) and then cultured at 30°C for 5 to 6 days. The colonies formed are purely isolated on the agar medium.
- The thus-obtained strains are cultured in the same manner as in Example 1 below and reacted with a lactonitrile. After completion of the reaction, the system is subjected to centrifugation to remove the microbial cells. The supernatant liquor is analyzed by liquid chromatography (column: SHODEX ODS F511A, carrier: 0.2M H₃PO₂, column temp.: 30°C, monitor wavelength: 208 nm) to determine an amount of lactic acid produced.
- In addition, its optical purity is determined in the same manner as in Example 1 below.
- Specific examples of microorganisms which can be used in the present invention include Enterobacter sp. SK12 (FERM No. BP-3322), Enterobacter taylorae JCM 3943, Arthrobacter sp. SK103 (FERM No. P-11300), Arthrobacter sp. HR1 (FERM No. BP-3323), Arthrobacter oxydans IFO 12138, Caseobacter sp. BC4 (FERM No. BP-3316), Caseobacter sp. BC23 (FERM No. P-11261), Brevibacterium acetylicum IAM 1790, Brevibacterium helvolum ATCC 11822, Aureobacterium testaceum IAM 1561, Escherichia coli IFO 3301, Micrococcus luteus ATCC 383, Micrococcus varians IAM 1099, Micrococcus roseus IFO 3768, Streptomyes griseus IFO 3355, Streptomyes griseus IFO 14059, Flavobacterium flavescens ATCC 8315, Flavobacterium sp. SK150 (FERM No. P-11645), Aeromonas punctata IFO 13288, Nocardia calcarea KCC A0191, Nocardia polychromogenes IFM 19, Mycoplana dimorpha ATCC 4279, Cellulomonas fimi IAM 12107, Erwinia herbicola IFO 12686, Candida guilliermondii IFO 0566, Candida lypolytica IAM 4964, Pseudomonas sp. SK13 (FERM No. BP-3325), Pseudomonas sp. SK31 (FERM No. P-11310), Pseudomonas sp. SK87 (FERM No. P-11311), Pseudomonas sp. BC13-2 (FERM No. BP-3319), Pseudomonas sp. BC15-2 (FERM No. BP-3320), Pseudomonas synxantha IAM 12356, Pseudomonas aeruginosa IAM 1267, Pseudomonas ovalis IAM 1002, Rhodococcus erythropolis IFO 12540, Rhodococcus erythropolis IFO 12320, Rhodococcus sp. SK92 (FERM No. BP-3324), Rhodococcus sp. HR11 (FERM No. P-11306), Rhodococcus sp. SK70 (FERM No. P-11304), Rhodococcus erythropolis IFO 12538, Rhodococcus erythropolis IFO 12539, Rhodococcus erythropolis IFM 132, Rhodococcus erythropolis IFM 155, Rhodococcus rhodochrous ATCC 12674, Bacillus licheniformis IFO 12197, Bacillus licheniformis IFO 12199, Bacillus megaterium ATCC 25833, Bacillus subtilis ATCC 21697, Alcaligenes sp. BC20 (FERM No. P-11264), Corynebacterium flavescens IAM 1642, Microbacterium lacticum IAM 1640 and Obsumbacterium proteus ATCC 12841, and variants of those strains.
- Of those strains, Arthrobacter oxydans IFO 12138, Brevibacterium acetylicum IAM 1790, Brevibacterium helvolum ATCC 11822, Aureobacterium testaceum IAM 1561, Escherichia coli IFO 3301, Micrococcus luteus ATCC 383, Micrococcus varians IAM 1099, Micrococcus roseus IFO 3768, Streptomyces griseus IFO 3355, Streptomyces griseus IFO 14059, Flavobacterium flavescens ATCC 8315, Aeromonas punctata IFO 13288, Nocardia calcarea KCC A0191, Nocardia polychromogenes IFM 19, Mycoplana dimorpha ATCC 4279, Cellulomonas fimi IAM 12107, Erwinia herbicola IFO 12686, Candida guilliermondii IFO 0566, Candida lypolytica IAM 4964, Pseudomonas synxantha IAM 12356, Pseudomonas aeruginosa IAM 1267, Pseudomonas ovalis IAM 1002, Rhodococcus erythropolis IFO 12540, Rhodococcus erythropolis IFO 12320, Rhodococcus erythropolis IFO 12538, Rhodococcus erythropolis IFO 12539, Rhodococcus erythropolis IFM 132, Rhodococcus erythropolis IFM 155, Rhodococcus rhodochrous ATCC 12674, Bacillus licheniformis IFO 12197 Bacillus licheniformis IFO 12199, Bacillus megaterium ATCC 25833, Bacillus subtilis ATCC 21697, Corynebacterium flavescens IAM 1642, Microbacterium lacticum IAM 1640 and Obsumacterium proteus ATCC 12841 are known strains that are available from the American Type Culture Collection (ATCC), the Institute for Fermentation, Osaka (IFO); the Research Institute for Chemobiodynamics, The University of Chiba (IFM); the Institute of Applied Microbiology, The University of Tokyo (IAM); the Tokyo Research Laboratories, Kaken Pharmaceutical Co., Ltd. (KCC); and the Japan Collection of Microorganisms, RIKEN (JCM) under the deposit number listed above.
- Other microorganisms are new strains isolated from nature by the present inventors and have been deposited with the Fermentation Research Institute, Agency of Industrial Science & Technology under the respective deposit number (FERM Nos.) listed above. The morphologic and physiologic properties of the new strains are described below.
-
- The above-described taxonomical properties were examined by referring to Bergey's Manual of Systematic Bacteriology (1986). As a result, the SK12 strain was identified to belong to the genus Enterobacter; the SK103 and HR1 strains to the genus Arthrobacter; the BC4 and BC23 strains to the genus Caseobacter; the SK150 strain to the genus Flavobacterium; the SK13, SK31, SK87, BC13-2 and BC15-2 strains to the genus Pseudomonas; the SK92, HR11 and SK70 strains to the genus Rhodococcus; and the BC20 strain to the genus Alcaligenes, respectively.
- The present invention is explained further below by referring to a generally adopted embodiment.
- Culture media which can be used for culturing the microorganisms according to the present invention usually contain assimilable carbon sources and nitrogen sources and inorganic nutrients necessary for growth. Carbon sources include glucose, glycerol and sucrose. Nitrogen sources include yeast extract, peptone and ammonium sulfate. Inorganic nutrients include dipotassium hydrogenphosphate, magnesium chloride and ferric chloride. It is preferable to obtain higher enzyme activity by adding nitriles (e.g., 2-chloropropionitrile, acetonitrile, propionitrile, n-butyronitrile, isobutyronitrile, benzonitrile, benzyl cyanide and 3-cyanopyridine) or, amides or lactams corresponding to those nitriles (e.g., ε-caprolactam and γ-butyrolactam) to the culture in the initial or middle stage of culturing in such a concentration that does not impair greatly growth of the microorganism.
- Culturing is conducted aerobically under controlled conditions selected according to the microorganism, i.e., at a pH of from 4 to 10 and at a temperature of from 20 to 90°C for a period of from 24 to 96 hours.
- The hydrolysis of DL-lactonitrile is carried out by bringing DL-lactonitrile into contact with the thus-obtained microbial cells or treated microbial cells (ruptured cells, crude or purified enzyme isolated therefrom, immobilized microbial cells or enzyme) in a polar solvent, whereby a desired optically active lactic acid, i.e., D- or L-lactic acid, is obtained from DL-lactonitrile in a predominant proportion (i.e., 50 to 100%) and in a high yield. Typical examples of usable polar solvents include water and an aqueous medium, e.g., physiologic saline and a phosphoric acid buffer solution. If desired, organic solvents, such as lower alcohols (e.g., methanol and ethanol), dimethylformamide and dioxane, may be used in combination with the polar solvent.
- The hydrolysis reaction also can be carried out by using acetaldehyde and prussic acid in place of DL-lactonitrile.
- DL-Lactonitrile usually is used at a concentration of from 0.01 to 10% by weight in the reaction system and the concentrations of acetaldehyde and prussic acid are 0.01 to 5% by weight and 0.005 to 3% by weight, respectively. The microbial cells usually are used in an amount of from 0.01 to 5% by weight (dry basis) based on DL-lactonitrile. The reaction is performed at a pH of from 3 to 12, and preferably from 6 to 10, and at a temperature of from the freezing point to 70°C, and preferably from 10 to 50°C for a period of from 0.5 to 72 hours.
- After the microbial cells are removed from the reaction system by centrifugation or the like means, the desired optically active lactic acid is isolated from the reaction solution by any of known techniques, such as concentration, electrical dialysis, ion exchange, extraction and crystallization.
- According to the present invention, a desired optically active lactic acid can be prepared directly from DL-lactonitrile in a predominant proportion (50 to 100%). The present invention makes it feasible to convert stoichiometrically all the raw material to a desired optically active lactic acid, thus providing an extremely efficient process for preparing optically active lactic acids.
- The present invention is now illustrated in greater detail with reference to the following Examples, but it should be understood that the present invention is not deemed to be limited thereto.
-
- Each of the microorganisms shown in Table 1 below was inoculated into 10 mℓ of a medium having the following composition in a test tube (diameter: 22 mm) and shake-cultured at 30°C for 2 days.
- The microbial cells were collected from each culture by centrifugation and washed with a 30 mM phosphoric acid buffer solution (pH=7.0). The precipitated cells were collected and resuspended in 5 mℓ of the same buffer solution. DL-Lactonitrile was added to the cell suspension in a final concentration of 10 mM, followed by incubation at 30°C for 24 hours. After completion of the reaction, the system was subjected to centrifugation to remove the microbial cells. The supernatant liquor was analyzed by column chromatography using a matrix, "MCI-GEL-CRS10W", produced by Mitsubishi Chemical Co., Ltd. or by an enzyme analysis using D-or L-lactic acid dehydrogenase and nicotinamide adenine dinucleotide to determine the optically active lactic acid produced and its optical purity. The results obtained are shown in Table 1.
- Enterobacter sp. SK12 was cultured in the same manner as in Example 1, except for using 100 mℓ of the medium in a 500 mℓ volume Erlenmeyer flask.
- To the microbial cells collected from the culture in the same manner as in Example 1 was added 50 mM phosphoric acid buffer solution to prepare a cell suspension having a cell concentration of 30 (OD 630 nm). To the cell suspension was added DL-lactonitrile to a final concentration of 20 mM, and the reaction progress was observed over time. A 2 mℓ sample of the reaction system was taken out each 1 hour to determine the amount of lactic acid produced in the same manner as in Example 1. The results obtained are shown in Table 2 below.
- Enterobacter sp. SK12 was cultured in the same manner as in Example 2.
- To the microbial cells collected from the culture in the same manner as in Example 2 was added 50 mM phosphoric acid buffer solution to prepare a cell suspension having a cell concentration of 30 (OD 630 nm). To the cell syspension was added acetaldehyde and prussic acid to a final concentration of 20 mM, respectively, and the reaction was carried out at 30°C for 5 hours. The amount of lactic acid produced and its optical purity were determined in the same manner as in Example 1. The results obtainedare shown in the Table 3 below.
- While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the
- scope thereof.
Claims (5)
- A process for preparing an optically active lactic acid comprising subjecting DL-lactonitrile to the action of a microorganism belonging to the genus Enterobacter, Arthrobacter , Caseobacter , Brevibacterium , Aureobacterium, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas , Nocardia , Mycoplana , Cellulomonas , Erwinia , Candida, Pseudomonas, Rhodococcus, Bacillus, Alcaligenes, Corynebacterium , Microbacterium or Obsumbacterium, or treated microbial cells thereof, which said microorganism is capable of stereospecifically hydrolyzing a nitrile in a polar solvent to produce directly D-lactic acid or L-lactic acid in a predominant proportion to said starting DL-lactonitrile.
- The process of Claim 1, wherein said microorganism has the properties of Enterobacter sp. SK12 (FERM No. BP-3322), Arthrobacter sp. SK103 (FERM No. P-11300), Arthrobacter sp. HR1 (FERM No. BP-3323), Caseobacter sp. BC4 (FERM No. BP-3316), Caseobacter sp. BC23 (FERM No. P-11261), Flavobacterium sp. SK150 (FERM No. P-11645), Pseudomonas sp. SK13 (FERM No. BP-3325), Pseudomonas sp. SK31 (FERM No. P-11310), Pseudomonas sp. SK87 (FERM No. P-11311), Pseudomonas sp. BC13-2 (FERM No. BP-3319), Pseudomonas sp. BC15-2 (FERM No. BP-3320), Rhodococcus sp. SK92 (FERM No. BP-3324), Rhodococcus sp. HR11 (FERM No. P-11306), Rhodococcus sp. SK70 (FERM No. P-11304) or Alcaligenes sp. BC20 (FERM No. P-11264), or a variant of those strains.
- The process of Claim 1, wherein said polar solvent is water, physiologic saline or a phosphoric acid buffer solution.
- A process for preparing an optically active lactic acid comprising subjecting acetaldehyde and prussic acid to the action of a microorganism belonging to the genus Enterobacter, Arthrobacter, Caseobacter, Brevibacterium, Aureobacterium, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Nocardia, Mycoplana, Cellulomonas, Erwinia, Candida, Pseudomonas, Rhodococcus, Bacillus, Alcaligenes, Corynebacterium, Microbacterium or Obsumbacterium, or treated microbial cells thereof, which said microorganism is capable of stereo specifically hydrolyzing a nitrile in a polar solvent to produce directly D-lactic acid or L-lactic acid in a predominant proportion to said acetaldehyde and prussic acid.
- The process of Claim 4, wherein said microorganism has the properties of Enterobacter sp. SK12 (FERM No. BP-3322), Arthrobacter sp. SK103 (FERM No. P-11300), Arthrobacter sp. HR1 (FERM No. BP-3323), Caseobacter sp. BC4 (FERM No. BP-3316), Caseobacter sp. BC23 (FERM No. P-11261), Flavobacterium sp. SK150 (FERM No. P-11645), Pseudomonas sp. SK13 (FERM No. BP-3325), Pseudomonas sp. SK31 (FERM No. P-11310), Pseudomonas sp. SK87 (FERM No. P-11311), Pseudomonas sp. BC13-2 (FERM No. BP-3319), Pseudomonas sp. BC15-2 (FERM No. BP-3320), Rhodococcus sp. SK92 (FERM No. BP-3324), Rhodococcus sp. HR11 (FERM No. P-11306), Rhodococcus sp. SK70 (FERM No. P-11304) or Alcaligenes sp. BC20 (FERM No. P-11264), or a variant of those strains.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2214916A JP2974737B2 (en) | 1990-08-16 | 1990-08-16 | Production method of optically active lactic acid |
| JP214916/90 | 1990-08-16 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| EP0473328A2 true EP0473328A2 (en) | 1992-03-04 |
| EP0473328A3 EP0473328A3 (en) | 1992-12-02 |
| EP0473328B1 EP0473328B1 (en) | 1997-06-25 |
Family
ID=16663707
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP91307462A Expired - Lifetime EP0473328B1 (en) | 1990-08-16 | 1991-08-13 | Biological process for preparing optically active lactic acid |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US5234826A (en) |
| EP (1) | EP0473328B1 (en) |
| JP (1) | JP2974737B2 (en) |
| DE (1) | DE69126646T2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0654533A2 (en) * | 1993-11-18 | 1995-05-24 | Mitsubishi Rayon Co., Ltd. | A process for producing D-lactic acid and L-lactamide |
| US6869783B1 (en) | 1998-10-19 | 2005-03-22 | Basf Aktiengesellschaft | Method for producing chiral carboxylic acids from nitriles with the assistance of a nitrilase or microoganisms which contain a gene for the nitrilase |
| US7985572B2 (en) | 2003-02-27 | 2011-07-26 | Basf Se | Modified nitrilases and their use in methods for the production of carboxylic acids |
| CN107190029A (en) * | 2016-03-14 | 2017-09-22 | 浦项工科大学校产学协力团 | Use the preparation of the D type lactic acid of novel enzyme |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3354688B2 (en) * | 1994-01-28 | 2002-12-09 | 三菱レイヨン株式会社 | Method for producing α-hydroxy acid or α-hydroxyamide by microorganism |
| DE69620847T3 (en) | 1995-11-10 | 2008-11-27 | Mitsubishi Rayon Co., Ltd. | Process for the preparation of an alpha-hydroxy acid or an alpha-hydroxyamide by means of microorganisms |
| WO1997032030A1 (en) * | 1996-02-29 | 1997-09-04 | Nippon Soda Co., Ltd. | PROCESS FOR PREPARING α-HYDROXY ACIDS USING MICROORGANISM AND NOVEL MICROORGANISM |
| US6037155A (en) * | 1997-02-27 | 2000-03-14 | Nippon Soda Co., Ltd. | Process for preparing α-hydroxy acids using microorganism and novel microorganism |
| US6416980B1 (en) | 2001-02-23 | 2002-07-09 | E. I. Du Pont De Nemours & Company | Method for producing glycolic acid from glycolonitrile using nitrilase |
| EP2198867A1 (en) * | 2001-12-07 | 2010-06-23 | Vertex Pharmaceuticals, Inc. | Pyrimidine-based compounds useful as GSK-3 inhibitors |
| US6582943B1 (en) | 2002-02-05 | 2003-06-24 | E. I. Du Pont De Nemours And Company | Method for producing 2-hydroxyisobutyric acid and methacrylic acid from acetone cyanohydrin |
| US7300787B2 (en) * | 2002-07-05 | 2007-11-27 | Archer-Daniels-Midland Company | Lactobacillus strains and use thereof in fermentation for L-lactic acid production |
| US7198927B2 (en) | 2004-12-22 | 2007-04-03 | E. I. Du Pont De Nemours And Company | Enzymatic production of glycolic acid |
| US20060160198A1 (en) * | 2004-12-22 | 2006-07-20 | Xu Li | Method for the production of glycolic acid from ammonium glycolate by direct deammoniation |
| US7445917B2 (en) * | 2004-12-22 | 2008-11-04 | E.I. Du Pont De Nemours And Company | Process for producing glycolic acid from formaldehyde and hydrogen cyanide |
| WO2006069127A1 (en) * | 2004-12-22 | 2006-06-29 | E.I. Dupont De Nemours And Company | Method for the production of glycolic acid from ammonium glycolate by solvent extraction |
| JP2009142256A (en) * | 2007-03-19 | 2009-07-02 | Sumitomo Chemical Co Ltd | Method for producing d-lactic acid |
| US7871802B2 (en) | 2007-10-31 | 2011-01-18 | E.I. Du Pont De Nemours And Company | Process for enzymatically converting glycolonitrile to glycolic acid |
| US7741088B2 (en) * | 2007-10-31 | 2010-06-22 | E.I. Dupont De Nemours And Company | Immobilized microbial nitrilase for production of glycolic acid |
| EP2460882B1 (en) * | 2009-07-28 | 2017-11-29 | Mitsui Chemicals, Inc. | Method for producing d-lactic acid |
| US8551535B1 (en) | 2012-08-06 | 2013-10-08 | Sarah McCann | Homeopathic remedies and methods for enhancing weight loss |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0187680A2 (en) * | 1985-01-11 | 1986-07-16 | Nitto Kagaku Kogyo Kabushiki Kaisha | Process for producing organic acids by use of microorganisms |
| EP0348901A2 (en) * | 1988-06-27 | 1990-01-03 | Asahi Kasei Kogyo Kabushiki Kaisha | Process for producing optically active alfa-substituted organic acid and microorganism and enzyme used therefor |
| EP0356912A2 (en) * | 1988-08-29 | 1990-03-07 | Idemitsu Kosan Company Limited | A method for the preparation of an optically active 2-substituted carboxylic acid |
| EP0486289A2 (en) * | 1990-11-14 | 1992-05-20 | Nitto Chemical Industry Co., Ltd. | Biological process for producing alpha-hydroxyamide or alpha-hydroxy acid |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2245585B1 (en) * | 1973-09-19 | 1976-05-14 | Anvar | |
| JPS6156086A (en) * | 1984-08-27 | 1986-03-20 | Asahi Chem Ind Co Ltd | Microbial preparation of alpha-hydroxyacid and its salt |
| JPS63222696A (en) * | 1987-03-13 | 1988-09-16 | Idemitsu Kosan Co Ltd | Production of alpha-hydroxycarboxylic acid |
-
1990
- 1990-08-16 JP JP2214916A patent/JP2974737B2/en not_active Expired - Lifetime
-
1991
- 1991-08-13 DE DE69126646T patent/DE69126646T2/en not_active Expired - Fee Related
- 1991-08-13 EP EP91307462A patent/EP0473328B1/en not_active Expired - Lifetime
- 1991-08-15 US US07/745,297 patent/US5234826A/en not_active Expired - Lifetime
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0187680A2 (en) * | 1985-01-11 | 1986-07-16 | Nitto Kagaku Kogyo Kabushiki Kaisha | Process for producing organic acids by use of microorganisms |
| EP0348901A2 (en) * | 1988-06-27 | 1990-01-03 | Asahi Kasei Kogyo Kabushiki Kaisha | Process for producing optically active alfa-substituted organic acid and microorganism and enzyme used therefor |
| EP0356912A2 (en) * | 1988-08-29 | 1990-03-07 | Idemitsu Kosan Company Limited | A method for the preparation of an optically active 2-substituted carboxylic acid |
| EP0486289A2 (en) * | 1990-11-14 | 1992-05-20 | Nitto Chemical Industry Co., Ltd. | Biological process for producing alpha-hydroxyamide or alpha-hydroxy acid |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0654533A2 (en) * | 1993-11-18 | 1995-05-24 | Mitsubishi Rayon Co., Ltd. | A process for producing D-lactic acid and L-lactamide |
| EP0654533A3 (en) * | 1993-11-18 | 1997-06-11 | Mitsubishi Rayon Co | A process for producing D-lactic acid and L-lactamide. |
| US6869783B1 (en) | 1998-10-19 | 2005-03-22 | Basf Aktiengesellschaft | Method for producing chiral carboxylic acids from nitriles with the assistance of a nitrilase or microoganisms which contain a gene for the nitrilase |
| US7985572B2 (en) | 2003-02-27 | 2011-07-26 | Basf Se | Modified nitrilases and their use in methods for the production of carboxylic acids |
| CN107190029A (en) * | 2016-03-14 | 2017-09-22 | 浦项工科大学校产学协力团 | Use the preparation of the D type lactic acid of novel enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0473328A3 (en) | 1992-12-02 |
| DE69126646D1 (en) | 1997-07-31 |
| US5234826A (en) | 1993-08-10 |
| DE69126646T2 (en) | 1997-10-23 |
| JPH0499497A (en) | 1992-03-31 |
| JP2974737B2 (en) | 1999-11-10 |
| EP0473328B1 (en) | 1997-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5234826A (en) | Biological process for preparing optically active lactic acid | |
| EP0486289B1 (en) | Biological process for producing alpha-hydroxyamide or alpha-hydroxy acid | |
| US5356812A (en) | Processes for production of optically active 3-phenyl-1,3-propanediol by asymmetric assimilation | |
| US5508181A (en) | Process for producing alpha-hydroxy acid or alpha-hydroxyamide by microorganisms | |
| CA1318871C (en) | Method for producing 2-keto-l-gulonic acid | |
| EP0449648B1 (en) | Process for producing R(-)-mandelic acid and derivatives thereof | |
| EP0188316B1 (en) | Process for the preparation of amides using microorganisms | |
| US5179014A (en) | Process for the preparation of amides using microorganisms | |
| US5200331A (en) | Method of producing an amide utilizing a microorganism | |
| EP0204555B1 (en) | Method of producing an amide utilizing a microorganism | |
| KR100339723B1 (en) | Process for production of amide compounds using microorganism | |
| US4943528A (en) | Process for the production of optically active (R)-(-)-3-halo-1,2-propanediol | |
| HU215093B (en) | Process for production of amide compounds | |
| JP3014171B2 (en) | Method for producing 4-halo-3-hydroxybutyramide | |
| US4933289A (en) | Biologically pure cultures of Pseudomonas sorbosoxidans useful for producing 2-keto-L-gulonic acid | |
| JP2696424B2 (en) | Method for producing R (-)-mandelic acid | |
| US5155030A (en) | Process for preparing optically active (R)-(-)-3-halo-1,2-propanediol from an epihalohydrin by a strain of corynebacterium or microbacterium | |
| US4892823A (en) | Method for producing 2-keto-L-gulonic acid | |
| US5258305A (en) | Manufacture of optically active 2-phenylpropionic acid and 2-phenylpropionamide from the nitrile using Rhodococcus equi | |
| US6645752B2 (en) | Microorganisms useful in a method of producing α-halo-α, β-saturated carbonyl compounds | |
| JP3090761B2 (en) | Production method of optically active lactic acid | |
| US5194380A (en) | Process for the production of optically active 2-hydroxy-4-phenyl-3-butenoic acid | |
| JP2983695B2 (en) | Method for producing 4-halo-3-hydroxybutyric acid | |
| US6465228B1 (en) | Levodione reductase | |
| JP4269416B2 (en) | Process for producing α-halo-α, β-saturated carbonyl compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): DE FR GB |
|
| PUAL | Search report despatched |
Free format text: ORIGINAL CODE: 0009013 |
|
| AK | Designated contracting states |
Kind code of ref document: A3 Designated state(s): DE FR GB |
|
| 17P | Request for examination filed |
Effective date: 19930526 |
|
| 17Q | First examination report despatched |
Effective date: 19960430 |
|
| GRAG | Despatch of communication of intention to grant |
Free format text: ORIGINAL CODE: EPIDOS AGRA |
|
| GRAH | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOS IGRA |
|
| GRAH | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOS IGRA |
|
| GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
| AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): DE FR GB |
|
| REF | Corresponds to: |
Ref document number: 69126646 Country of ref document: DE Date of ref document: 19970731 |
|
| ET | Fr: translation filed | ||
| PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
| 26N | No opposition filed | ||
| REG | Reference to a national code |
Ref country code: FR Ref legal event code: TP |
|
| REG | Reference to a national code |
Ref country code: GB Ref legal event code: 732E |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20000807 Year of fee payment: 10 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20000809 Year of fee payment: 10 |
|
| PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20000811 Year of fee payment: 10 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GB Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20010813 |
|
| GBPC | Gb: european patent ceased through non-payment of renewal fee |
Effective date: 20010813 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FR Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20020430 |
|
| PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20020501 |
|
| REG | Reference to a national code |
Ref country code: FR Ref legal event code: ST |